Retroviruses induce cancer and immunodeficiency diseases in both humans and animals. Some commonly known human retroviruses are HIV-1 and human T-lymphotropic virus type I. Our laboratory uses Rous sarcoma virus (RSV), an oncogenic avian retrovirus, as a model system to study viral trafficking and pathogen- host interactions. The major structural protein of retroviruses, Gag, directs the encapsidation of genomic viral RNA into virus particles during assembly. Retroviral assembly occurs at the plasma membrane; however, our laboratory has shown that the RSV Gag protein transiently traffics through the nucleus. It was found that nuclear trafficking of RSV Gag is linked to efficiet packaging of genomic RNA, suggesting that Gag may bind the RNA genome in the nucleus. Through studies performed with prototype foamy virus and murine leukemia virus, it was discovered the Gag was able to interact with host chromatin during proviral integration. The hypothesis of this proposal is that RSV Gag tethers to chromatin or chromatin-bound proteins as a strategy for capturing the newly transcribed viral genomic RNA. The goal of this proposal is to delineate the role of RSV Gag interaction with host chromatin-associated factors. Using the techniques of biochemical fractionation, chromatin immunoprecipitation, and immunofluorescence, it will be determined whether RSV Gag associates with chromatin or chromatin-bound proteins. In unpublished work, we found that RSV Gag is present in chromatin-associated protein fractions when subcellular fractionations were performed. To determine whether this phenomenon is conserved among other retroviruses, subcellular fractionations will be performed on a variety of Gag proteins from different retroviral genera. To identify Gag-associated host proteins, proteomic studies using affinity purification with affinity tagged RSV Gag and nuclear lysates were performed. Included in this proteomic analysis were proteins associated with the mediator complex, which is necessary for RNA polymerase II transcription. To determine whether mediator proteins have a crucial role in RSV genomic RNA packaging, mediator knockdown and overexpression experiments will be performed. Effects on viral genomic RNA packaging will be determined through RT-PCR of the RNA within virus particles collected after mediator knockdown or overexpression. To further identify other chromatin proteins that could interact with RSV Gag, tandem affinity purification experiments will be performed. These studies will shed some light into the early steps of retroviral genome recognition, possibly leading to the identification of future targets for drug therapies.